1. Field of the Invention
The present invention relates, in general, to torsin genes, preferably, torsina which encodes the torsion dystonia gene, DYT1. In particular, the present invention relates to nucleic acid molecules coding for the torsin protein; purified torsin proteins and polypeptides; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antibodies having binding affinity specifically to torsin proteins and polypeptides; hybridomas containing the antibodies; nucleic acid probes for the detection of nucleic acids encoding torsin proteins; a method of detecting nucleic acids encoding torsin proteins or polypeptides in a sample; kits containing nucleic acid probes or antibodies; bioassays using the nucleic acid sequence, protein or antibodies of this invention to diagnose, assess, or prognose a mammal afflicted with torsion dystonia; therapeutic uses; and methods of preventing torsion dystonia in an animal (preferably, a human).
2. Related Art
Movement disorders constitute a group of human neurologic diseases in which aberrant neurotransmission in the basal ganglia is associated with uncontrollable body movements, such as chorea in Huntington disease, tremor and rigidity in Parkinson disease, and twisting contraction in torsion dystonia. Dystonic symptoms can be secondary to a number of neurologic conditions, and to drug or traumatic injury to the brain, but primary or torsion dystonia is distinguished by lack of other neurologic involvement (Fahn, S., Adv Neurol 50:1-8 (1988); Chutorian, A. H., Acta Neuropediatricia 2:33-45 (1996)) and, in contrast to these other two neurodegenerative diseases, the absence of any distinct neuropathology. The clinical manifestations of dystonia show wide variations in age and site of onset, as well as body regions involved. The prevalence of all forms of primary dystonia is estimated at 3/10,000 in North America (Nutt, J. G., et al., Mov Disord 3:188-194 (1988)).
Early onset, generalized dystonia is the most disabling form of primary dystonia. Symptoms usually begin in an arm or leg at around 12 yrs (range 4-44 years) and spread to involve other limbs within about 5 years (Bressman, S. B., et al., Annal Neurol 36:771-777 (1994b); Greene, P., et al., Mov Disord 10:143-152 (1995)). The clinical spectrum of early onset dystonia is similar in all ethnic populations, with highest prevalence in the Ashkenazi Jewish (termed here AJ) population (Zeman, W., and Dyken, P., Psychiatr Neurol Neurochir 10:77-121 (1967); Korczyn, A. D., et al., Ann Neurol 8:387-391 (1980); Eldridge, R., Neurology 20:1-78 (1970)), due to a founder mutation (Ozelius, L., et al., Am. J. Hum. Genet. 50:619-628 (1992); Risch, N. J., et al., Nature Genetics 9:152-159 (1995)). Early onset dystonia follows an autosomal dominant mode of inheritance with 30-40% penetrance (Bressman, S. B., et al., Ann Neurol 26:612-620 (1989); Risch, N. J., et al., Am J Hum Genet 46:533-538 (1990)). The responsible gene in Jewish and non-Jewish families has been mapped to human chromosome 9q34 (Ozelius, L., et al., Neuron 2:1427-1434 (1989); Kramer, P. L., et al., Ann Neurol 27:114-120 (1990) and Kramer, P., et al., Am J Hum Gen 55:468-475 (1994)). Haplotype analysis of the founder mutation in AJ families placed the DYT1 gene in a 1-2 cM interval centromeric to the ASS locus on chromosome 9 (Ozelius, L., et al., Am. J. Hum. Genet. 50:619-628 (1992)) with highest lod scores obtained with adjacent markers, D9S62a/b and D9S63 (Risch, N., et al., Nature Genetics 9:152-159 (1985)).
The present invention relates to dystonia, dystonia genes and encoded proteins and mutations in dystonia genes that result in a dystonia disorder. In particular, the invention provides isolated nucleic acid molecules coding for torsin, preferably, torsinA which encodes the torsion dystonia gene, DYT1.
The invention further provides purified polypeptides comprising amino acid sequences encoding torsin proteins.
The invention also provides nucleic acid probes for the specific detection of the presence of and mutations in nucleic acids encoding torsin proteins or polypeptides in a sample.
The invention further provides a method of detecting the presence of and mutations in a nucleic acid encoding torsin protein in a sample.
The invention also provides a kit for detecting the presence of and mutations in a nucleic acid encoding torsin protein in a sample.
The invention further provides a recombinant nucleic acid molecule comprising, 5xe2x80x2 to 3xe2x80x2, a promoter effective to initiate transcription in a host cell and the above-described isolated nucleic acid molecule.
The invention also provides a recombinant nucleic acid molecule comprising a vector and the above-described isolated nucleic acid molecule.
The invention further provides a recombinant nucleic acid molecule comprising a sequence complimentary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide.
The invention also provides a cell that contains the above-described recombinant nucleic acid molecule.
The invention further provides a non-human organism that contains the above-described recombinant nucleic acid molecule.
The invention also provides an antibody having binding affinity specifically to a torsin protein or polypeptide.
The invention further provides a method of detecting torsin protein or polypeptide in an sample.
The invention also provides a method of measuring the amount of torsin protein or polypeptide in a sample.
The invention further provides a method of detecting antibodies having binding affinity specifically to a torsin protein or polypeptide.
The invention further provides a diagnostic kit comprising a first container means containing a conjugate comprising a binding partner of the monoclonal antibody and a label.
The invention also provides a hybridoma which produces the above-described monoclonal antibody.
The invention further provides diagnostic methods for dystonia disorders in humans, in particular, torsion dystonia. Preferably, a method of diagnosing the presence or absence of dystonia; predicting the likelihood of developing or a predisposition to develop dystonia in a human is provided herein. Specifically, methods of the present invention encompass detecting the presence, or absence of, a mutation in a gene wherein the mutation results in a dystonia disorder that affects humans. For example, the method comprises obtaining a sample from a human patient; evaluating the characteristics of torsinA nucleic acid in the sample, wherein the evaluation comprises detecting the GAGGAG region (SEQ ID NO: 5 at nucleotide positions 946-951) in the sample; and diagnosing the presence or predisposition to develop torsion dystonia in a patient wherein the absence of three nucleotides from the GAGGAG region indicates the presence or predisposition to develop torsion dystonia.
The present invention also encompasses methods for diagnosing the presence or absence of a dystonia disorder in a human comprising detecting the presence or absence of at least one mutation in a dystonia gene, wherein the presence of a mutation in the dystonia gene is indicative of a positive diagnosis and the absence of the mutation is indicative of the absence of a dystonia disorder. The dystonia disorder can be, for example, torsion dystonia. A biological sample obtained from a human can be used in the diagnostic methods. The biological sample can be a bodily fluid sample such as blood, saliva, semen, vaginal secretion, cerebrospinal and amniotic bodily fluid sample. Alternatively or additionally, the biological sample is a tissue sample such as a chorionic villus, neuronal, epithelial, muscular and connective tissue sample. In both bodily fluid and tissue samples, nucleic acids are present in the samples. In another embodiment the sample is a nucleic acid preparation obtained from human chromosome 9q34.
The dystonia gene can be the DYT1 gene (SEQ ID NO: 1). In normal humans (humans who are not affected by a dystonia disorder such as torsion dystonia) two normal alleles of the DYT1 gene are present. Humans affected with a dystonia disorder, such as torsion dystonia, have one normal allele and one abnormal allele characterized by at least one mutation in the nucleotide sequence. In one embodiment the mutation is a deletion mutation. Alternatively the mutation can be a missense, or frame shift mutation. In a preferred embodiment the deletion mutation is a deletion of one or more nucleotides from the GAGGAG region of SEQ ID NO: 5 at nucleotide positions 946-948; 949-951; 947-949; 948-950; or any combination thereof. For example, if the mutation to be detected is a deletion mutation, the presence or absence of three nucleotides in this region can result in the deletion of an A and two Gs, which, in turn, results in GAG rather than GAGGAG in the sequence. The presence or absence of these three nucleotides is indicative of a negative or positive diagnosis, respectively. The biological samples obtained from humans are evaluated in parallel to control samples with and without the appropriate dystonia disorder mutation.
The invention also relates to methods of detecting the presence or absence of dystonia disorder in a human wherein the dystonia disorder is characterized by one or more mutations in the dystonia gene. In this aspect of the invention a test sample comprising a dystonia gene is analyzed for the presence or absence of one or more mutations in the dystonia gene and compared to results of analysis of control samples. The test samples comprise biological samples from the human (e.g., blood, tissue). The control samples comprise biological samples with or without a mutation in the dystonia gene. The presence or absence of a mutation in the test sample is indicative of a positive or negative diagnosis, respectively, for a dystonia disorder.
Another aspect of the invention relates to methods of detecting the presence or absence of a dystonia disorder, wherein the test sample from the human is evaluated by performing a polymerase chain reaction with oligonucleotide primers capable of amplifying a dystonia gene, such as the DYT1 gene (SEQ ID NO: 1). The PCR specific primers can be, for example, designed for a region of exon 5 of the DYT1 gene (SEQ ID NO: 27), such as SEQ ID NOS: 28 and 29. Following PCR amplification of a nucleic acid sample, the amplified nucleic acid fragments are separated and mutations in the DYT1 gene and alleles of the dystonia gene detected. For example, a mutation in the DYT1 gene is indicative of the presence of the torsion dystonia, whereas the lack of a mutation is indicative of a negative diagnosis. In one embodiment the mutation is a deletion mutation comprising the deletion of three nucleotides in the DYT1 gene. In another embodiment the mutation is in three nucleotides from a GAGGAG region of SEQ ID NO: 5, preferably at nucleotide positions 946-948; 949-951; 947-949; 948-950; or any combination thereof. In yet another embodiment, the method further comprises the additional step of sequencing the amplified DNA fragments.
An additional aspect of the invention is a method of determining the presence or absence of a dystonia disorder in a human comprises the steps of contacting a biological sample obtained from the human with a nucleic acid probe to a dystonia gene; maintaining the biological sample and the nucleic acid probe under conditions suitable for a hybridization; detecting the hybridization between the biological sample and the nucleic acid probe; and comparing the hybridization signal obtained from the human to a control sample which does or does not contain a dystonia disorder. The absence of a hybridization signal is indicative of a positive diagnosis. The presence of a hybridization signal is indicative of a negative diagnosis. The dystonia disorder can be, for example, torsion dystonia. The hybridization is performed with a nucleic acid fragment of a dystonia gene such as DYT1 (SEQ ID NO: 1). The nucleic acid probe can be labeled (e.g., fluorescent, radioactive, enzymatic, biotin label).
The invention also encompasses methods for predicting whether a human is likely to be affected with a dystonia disorder, comprising obtaining a biological sample from the human; contacting the biological sample with a nucleic acid probe; maintaining the biological sample and the nucleic acid probe under conditions suitable for hybridization; and detecting the hybridization between the biological sample and the nucleic acid probe. In another embodiment the method further comprises performing a polymerase chain reaction with oligonucleotide primers capable of amplifying a dystonia gene (e.g., DYT1, SEQ ID NO: 1); and detecting amplified DNA fragments of the dystonia gene for a mutation, wherein the mutation in the dystonia gene is indicative of the presence or absence of the torsion dystonia. The hybridization can, for example, detect a deletion in nucleotides indicative of a positive diagnosis; or the presence of nucleotides indicative of a negative diagnosis. In one embodiment the nucleotides are a GAG from a GAGGAG region (SEQ ID NO: 5) for detecting torsion dystonia. The deletion mutation can be a deletion of nucleotides 946-948; 949-951; 947-949; 948-950; or any combination thereof from SEQ ID NO: 5. In another embodiment, the amplified DNA fragments can be sequenced to detect the presence or absence of mutations.
The invention further provides for methods for determining the presence or absence of a dystonia disorder in a human comprising obtaining a biological sample from the human; and assessing the level of a dystonia protein in the biological sample comprising bodily fluids, tissues or both from the human. The levels or concentrations of the dystonia protein are determined by contacting the sample with at least one antibody specific to the dystonia protein, and detecting the levels of the dystonia protein. An alteration in the dystonia protein levels is indicative of a diagnosis. The dystonia protein detected can be, for example, torsin A (SEQ ID NO: 2) encoded by the nucleic acid sequence of SEQ ID NO: 1. The antibody used in the method can be a polyclonal antibody or a monoclonal antibody and can be detectably labeled (e.g., fluorescence, biotin, colloidal gold, enzymatic).
In another embodiment the method of assessing the level or concentration of the dystonia protein further comprises contacting the sample with a second antibody specific to the dystonia protein or a complex between an antibody and the dystonia protein.
The present invention also provides for a kit for diagnosing the presence or absence of a dystonia disorder in a human comprising one or more reagents for detecting a mutation in a dystonia gene, such as DYT1, or a dystonia protein, such as torsin A, in a sample obtained from the human. The one or more reagents for detecting the torsion dystonia are used for carrying out an enzyme-linked immunosorbent assay or a radioimmunoassay to detect the presence of absence of dystonia protein. In another embodiment the kit comprises one or more reagents for detecting the torsion dystonia by carrying out a PCR, hybridization or sequence based assays or any combination thereof.
It is also envisioned that the methods of the present invention can diagnosis a mutation in a dystonia gene, such as DYT1, which encodes a dystonia protein, such as torsin A, wherein a mutation in the dystonia gene for the human is compared to a mutation in a dystonia gene for a parent of the human who is unaffected by a torsion dystonia, a parent of the human who is affected by the torsion dystonia and a sibling of the human who is affected by the torsin dystonia.
The invention also provides methods for therapeutic uses involving all or part of (1) the nucleic acid sequence encoding torsin protein or (2) torsin protein.
Further objects and advantages of the present invention will be clear from the description that follows.
In the description that follows, a number of terms used in recombinant DNA (rDNA) technology are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Isolated Nucleic Acid Molecule. An xe2x80x9cisolated nucleic acid moleculexe2x80x9d, as is generally understood and used herein, refers to a polymer of nucleotides, and includes but should not be limited to DNA and RNA. The xe2x80x9cisolatedxe2x80x9d nucleic acid molecule is purified from its natural in vivo state.
Recombinant DNA. Any DNA molecule formed by joining DNA segments from different sources and produced using recombinant DNA technology (aka. molecular genetic engineering).
DNA Segment. A DNA segment, as is generally understood and used herein, refers to a molecule comprising a linear stretch of nucleotides wherein the nucleotides are present in a sequence that can encode, through the genetic code, a molecule comprising a linear sequence of amino acid residues that is referred to as a protein, a protein fragment or a polypeptide.
Gene. A DNA sequence related to a single polypeptide chain or protein, and as used herein includes the 5xe2x80x2 and 3xe2x80x2 untranslated ends. The polypeptide can be encoded by a full-length sequence or any portion of the coding sequence, so long as the functional activity of the protein is retained.
Complementary DNA (cDNA). Recombinant nucleic acid molecules synthesized by reverse transcription of messenger RNA (xe2x80x9cmRNA).
Structural Gene. A DNA sequence that is transcribed into mRNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide.
Restriction Endonuclease. A restriction endonuclease (also restriction enzyme) is an enzyme that has the capacity to recognize a specific base sequence (usually 4, 5, or 6 base pairs in length) in a DNA molecule, and to cleave the DNA molecule at every place where this sequence appears. For example, EcoRI recognizes the base sequence GAATTC/CTTAAG.
Restriction Fragment. The DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome can be digested by a particular restriction endonuclease into a discrete set of restriction fragments.
Agarose Gel Electrophoresis. To detect a polymorphism in the length of restriction fragments, an analytical method for fractionating double-stranded DNA molecules on the basis of size is required. The most commonly used technique (though not the only one) for achieving such a fractionation is agarose gel electrophoresis. The principle of this method is that DNA molecules migrate through the gel as though it were a sieve that retards the movement of the largest molecules to the greatest extent and the movement of the smallest molecules to the least extent. Note that the smaller the DNA fragment, the greater the mobility under electrophoresis in the agarose gel.
The DNA fragments fractionated by agarose gel electrophoresis can be visualized directly by a staining procedure if the number of fragments included in the pattern is small. The DNA fragments of genomes can be visualized successfully. However, most genomes, including the human genome, contain far too many DNA sequences to produce a simple pattern of restriction fragments. For example, the human genome is digested into approximately 1,000,000 different DNA fragments by EcoRI. In order to visualize a small subset of these fragments, a methodology referred to as the Southern hybridization procedure can be applied.
Southern Transfer Procedure. The purpose of the Southern transfer procedure (also referred to as blotting) is to physically transfer DNA fractionated by agarose gel electrophoresis onto a nitrocellulose filter paper or another appropriate surface or method, while retaining the relative positions of DNA fragments resulting from the fractionation procedure. The methodology used to accomplish the transfer from agarose gel to nitrocellulose involves drawing the DNA from the gel into the nitrocellulose paper by capillary action.
Nucleic Acid Hybridization. Nucleic acid hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the thermodynamically favored double-stranded structure if they are mixed under the proper conditions. The double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a nitrocellulose filter. In the Southern hybridization procedure, the latter situation occurs. As noted previously, the DNA of the individual to be tested is digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, converted to the single-stranded form, and transferred to nitrocellulose paper, making it available for reannealing to the hybridization probe. Examples of hybridization conditions can be found in Ausubel, F. M. et al., Current protocols in Molecular Biology, John Wily and Sons, Inc., New York, N.Y. (1989). A nitrocellulose filter is incubated overnight at 68xc2x0 C. with labeled probe in a solution containing 50% formamide, high salt (either 5xc3x97SSC[20xc3x97: 3M NaCl/0.3M trisodium citrate] or 5xc3x97SSPE[20xc3x97: 3.6M NaCl/0.2M NaH2PO4/0.02M EDTA, pH 7.7]), 5xc3x97Denhardt""s solution, 1% SDS, and 100 xcexcg/ml denatured salmon sperm DNA. This is followed by several washes in 0.2xc3x97SSC/0.1% SDS at a temperature selected based on the desired stringency: room temperature (low stringency), 42xc2x0 C. (moderate stringency) or 68xc2x0 C. (high stringency). The temperature is selected is determined based on the melting temperature (Tm) of the DNA hybrid.
Hybridization Probe. To visualize a particular DNA sequence in the Southern hybridization procedure, a labeled DNA molecule or hybridization probe is reacted to the fractionated DNA bound to the nitrocellulose filter. The areas on the filter that carry DNA sequences complementary to the labeled DNA probe become labeled themselves as a consequence of the reannealing reaction. The ears of the filter that exhibit such labeling are visualized. The hybridization probe is generally produced by molecular cloning of a specific DNA sequence.
Oligonucleotide or Oligomer. A molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. AN oligonucleotide can be derived synthetically or by cloning.
Sequence Amplification. A method for generating large amounts of a target sequence. In general, one or more amplification primers are annealed to a nucleic acid sequence. Using appropriate enzymes, sequences found adjacent to, or in between the primers are amplified.
Amplification Primer. An oligonucleotide which is capable of annealing adjacent to a target sequence and serving as an initiation point for DNA synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is initiated.
Vector. A plasmid or phage DNA or other DNA sequence into which DNA can be inserted to be cloned. The vector can replicate autonomously in a host cells, and can be further characterized by one or a small number of endonuclease recognition sites at which such DNA sequences can be cut in a determinable fashion and into which DNA can be inserted. The vector can further contain a marker suitable for use in the identification of cells transformed with the vector. Markers, for example, are tetracycline resistance or ampicillin resistance. The words xe2x80x9ccloning vehiclexe2x80x9d are sometimes used for xe2x80x9cvector.xe2x80x9d
Expression. Expression is the process by which a structural gene produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptides(s).
Expression Vector. A vector or vehicle similar to a cloning vector but which is capable of expressing a gene which has been cloned into it after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and can additional contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
Functional Derivative. A xe2x80x9cfunctional derivativexe2x80x9d of a sequence, either protein or nucleic acid, is a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of the protein or nucleic acid sequence. A functional derivative of a protein can contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modification for the performance of a specific function. The term xe2x80x9cfunctional derivativexe2x80x9d is intended to include the xe2x80x9cfragment,xe2x80x9d xe2x80x9csegments,xe2x80x9d xe2x80x9cvariants,xe2x80x9d xe2x80x9canalogs,xe2x80x9d or xe2x80x9cchemical derivativesxe2x80x9d of a molecule.
As used herein, a molecule is said to be a xe2x80x9cchemical derivativexe2x80x9d of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule""s solubility, absorption, biological half life, and the like. The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed in Remington""s Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.
Variant. A xe2x80x9cvariantxe2x80x9d of a protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the protein or nucleic acid. Thus, provided that two molecules possess a common activity and can substitute for each other, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical.
Allele. An xe2x80x9callelexe2x80x9d is an alternative form of a gene occupying a given locus on the chromosome.
Mutation. A xe2x80x9cmutationxe2x80x9d is any detectable change in the genetic material which can be transmitted to daughter cells and possibly even to succeeding generations giving rise to mutant cells or mutant individuals. If the descendants of a mutant cell give rise only to somatic cells in multicellular organisms, a mutant spot or area of cells arises. Mutations in the germ line of sexually reproducing organisms can be transmitted by the gametes to the next generation resulting in an individual with the new mutant condition in both its somatic and germ cells. A mutation can be any (or a combination of) detectable, unnatural change affecting the chemical or physical constitution, mutability, replication, phenotypic function, or recombination of one or more deoxyribonucleotides; nucleotides can be added, deleted, substituted for, inverted, or transposed to new positions with and without inversion. Mutations can occur spontaneously and can be induced experimentally by application of mutagens. A mutant variation of a nucleic acid molecule results from a mutation. A mutant polypeptide can result form a mutant nucleic acid molecule and also refers to a polypeptide which is modified at one, or more, amino acid residues from the wildtype (naturally occurring) polypeptide. The term xe2x80x9cmutationxe2x80x9d, as used herein, can also refer to any modification in a nucleic acid sequence encoding a dystonia protein. For example, the mutation can be a point mutation or the addition, deletion, insertion and/or substitution of one or more nucleotides or any combination thereof. The mutation can be a missense or frameshift mutation. Modifications can be, for example, conserved or non-conserved, natural or unnatural.
Species. A xe2x80x9cspeciesxe2x80x9d is a group of actually or potentially interbreeding natural populations. A species variation within a nucleic acid molecule or protein is a change in the nucleic acid or amino acid sequence that occurs among species and can be determined by DNA sequencing of a molecule in question.
Polyacrylamide Gel Electrophoresis (PAGE). The most commonly used technique (though not the only one) for achieving a fractionation of polypeptides on the basis of size is polyacrylamide gel electrophoresis. The principle of this method is that polypeptide molecules migrate through the gel as though it were a sieve that retards the movement of the largest molecules to the greatest extent and the movement of the smallest molecules to the least extent. Note that the smaller the polypeptide fragment, the greater the mobility under electrophoresis in the polyacrylamide gel. Both before and during electrophoresis, the polypeptides typically are continuously exposed to the detergent sodium dodecyl sulfate (SDS), under which conditions the polypeptides are denatured. Native gels are run in the absence of SDS. The polypeptides fractionated by polyacrylamide gel electrophoresis can be visualized directly by a staining procedure if the number of polypeptide component is small.
Western Transfer Procedure. The purpose of the Western transfer procedure (also referred to as blotting) is to physically transfer polypeptides fractionated by polyacrylamide gel electrophoresis onto a nitrocellulose filter paper or another appropriate surface or method, while retaining the relative positions of polypeptides resulting from the fractionation procedure. The blot is then probed with an antibody that specifically binds to the polypeptide of interest.
Purified. A xe2x80x9cpurifiedxe2x80x9d protein or nucleic acid is a protein or nucleic acid that has been separated from a cellular component. xe2x80x9cPurifiedxe2x80x9d proteins or nucleic acids have been purified to a level of purity not found in nature.
Substantially Pure. A xe2x80x9csubstantially purexe2x80x9d protein or nucleic acid is a protein or nucleic acid preparation that is lacking in all other cellular components.
Nucleic Acids. Nucleic acids are defined herein as heteropolymers of nucleic acid molecules. Nucleic acid molecules are meant to refer to chains of nucleotides joined together by phosphodiester bonds to form a nucleic acid heteropolymer. The nucleic acid molecules can be double stranded or single stranded and can be deoxyribonucleotide (DNA) molecules, such as cDNA or genomic DNA, or ribonucleotide (RNA) molecules. As such, the nucleic acid molecule can include one or more exons, with or without, as appropriate, introns.
PCR. PCR refers to the polymerase chain reaction, a rapid procedure for the in vitro enzymatic amplification of nucleic acids. The nucleic acid to be amplified is denatured by heating in the presence of DNA polymerase, excess deoxyribonucleotide triphosphates and oligonucleotides that specifically hybridize to target sequences to prime new DNA synthesis. The amplification procedure is characterized by cycling which leads to a multi-fold amplification of a nucleic acid fragment of interest.